Domain mutagenesis kit随机突变试剂盒说明书

GeneMorph II EZClone Domain Mutagenesis KitINSTRUCTION MANUALCatalog #200552Revision C.01For In Vitro Use Only200552-12L IMITED P RODUCT W ARRANTYThis warranty limits our liability to replacement of this product. No other warranties of any kind,express or implied, including without limitation, implied warranties of merchantability or fitness fora particular purpose, are provided by Agilent. Agilent shall have no liability for any direct, indirect,consequential, or incidental damages arising out of the use, the results of use, or the inability to usethis product.O RDERING I NFORMATION AND T ECHNICAL S ERVICESUnited States and CanadaAgilent TechnologiesStratagene Products Division11011 North Torrey Pines RoadLa Jolla, CA 92037373-6300Telephone(858)424-5444Order Toll Free (800)Technical Services(800) 894-1304Internet techservices@World Wide Web EuropeServices Location Telephone Fax TechnicalAustria 0800 292 499 0800 292 496 0800 292 498Belgium00800 7000 7000 00800 7001 7001 00800 7400 74000800 15775 0800 15740 0800 15720France00800 7000 7000 00800 7001 7001 00800 7400 74000800 919 288 0800 919 287 0800 919 289 Germany00800 7000 7000 00800 7001 7001 00800 7400 74000800 182 **** **** 182 **** **** 182 8234 Netherlands00800 7000 7000 00800 7001 7001 00800 7400 74000800 023 0446 +31 (0)20 312 5700 0800 023 0448 Switzerland00800 7000 7000 00800 7001 7001 00800 7400 74000800 563 080 0800 563 082 0800 563 081 United Kingdom00800 7000 7000 00800 7001 7001 00800 7400 74000800 917 3282 0800 917 3283 0800 917 3281 All Other CountriesPlease contact your local distributor. A complete list of distributors is available at .C ONTENTSMaterials Provided (1)Storage Conditions (1)Additional Materials Required (1)Notice to Purchaser (2)Introduction (3)Eliminate Bias and Easily Control Mutation Rate (5)EZClone Reaction (9)GeneMorph II EZClone Domain Mutagenesis Control (10)XL10-Gold Ultracompetent Cells (10)Preprotocol Considerations (11)Mutant Megaprimer Synthesis Considerations (11)EZClone Reaction Considerations (13)Protocol (14)Mutant Megaprimer Synthesis (14)EZClone Reaction (17)Expected Results (21)Transformation Guidelines (22)Troubleshooting (23)Preparation of Media and Reagents (25)Appendix: How to Calculate Mutation Frequency (26)References (28)MSDS Information (28)Quick-Reference Protocol (29)M ATERIALS P ROVIDEDQuantity Materials provided a ConcentrationUU/μl 25 Mutazyme II DNA polymerase b 2.510× Mutazyme II reaction buffer 10× 150 μl40 mM dNTP mix c10 mM each dNTP 10 μl2× EZClone enzyme mix 2× 250 μlU Dpn I restriction enzyme 10 U/μl 100EZClone solution — 30 μlng Positive control plasmid 10 ng/μl 210ng Positive control primer mix 250 ng/μl 750μl1.1-kb gel standard 20 ng/μl 50XL10-Gold ultracompetent cells d,e (yellow tubes) — 4 × 135 μlXL10-Gold β-mercaptoethanol mix (β-ME) — 50μlng/μl 10μlpUC18 control plasmid (0.1 ng/μl in TE buffer f) 0.1a Sufficient reagents are provided for 10 reactions, which includes 3 control reactions.b Mutazyme II DNA polymerase is not sold separately.c Thaw the dNTP mix once, prepare single-use aliquots, and store the aliquots at –20°C. Do not subject the dNTP mixto multiple freeze-thaw cycles.d Genotype: Tet rΔ (mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte[F’ proAB lacI q ZΔM15 Tn10 (Tet r) Amy Cam r]e The XL10-Gold ultracompetent cells must be stored at the bottom of a –80°C freezer immediately on receipt.The ultracompetent cells are very sensitive to small variations in temperature. Transferring tubes from one freezer toanother may result in a loss of efficiency.f The pUC18 control plasmid is stored in TE buffer (see Preparation of Media and Reagents).S TORAGE C ONDITIONSXL10-Gold ultracompetent cells: Store the cells immediately at –80°C.Do not place the cells in liquid nitrogen.All other components: Store at –20°C upon receipt. Store the 2× EZClone enzyme mix at4°C after thawing. Once thawed, full activity is guaranteed for 3 months.A DDITIONAL M ATERIALS R EQUIREDTemperature cyclerPCR tubes llPCR primers14-ml BD Falcon polypropylene round-bottom tubes (BD Biosciences Catalog #352059)ll Thin-walled PCR tubes are highly recommended for use with Stratagene thermal cyclers. These PCR tubes ensure idealcontact with the multiblock design to permit more efficient heat transfer and to maximize temperature cyclingperformance.Revision C.01 © Agilent Technologies, Inc. 2009.N OTICE TO P URCHASERNotice to Purchaser: Limited LicensePurchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser’s own internal research. No other patent rights (such as 5’ Nuclease Process patent rights) are conveyed expressly, by implication, or by estoppel.Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.I NTRODUCTIONRandom mutagenesis is a powerful tool for elucidating protein structure-function relationships and for modifying proteins to improve or alter their characteristics. E rror-prone PCR is a random mutagenesis technique for generating amino acid substitutions in proteins, domains or promoter elements by introducing mutations during PCR. However, cloning amplicons generated by error-prone PCR can be difficult and labor-intensive due to low product yields, mutations at the ends which interfere with restriction-based cloning, and/or inefficient synthesis of 3´ dA overhangs or blunt ends which reduces the efficiency of TA- or blunt-end cloning strategies. In addition, targeting specific functional domains for random mutagenesis can also be difficult and inefficient using current methods. To address the need for efficient and flexible cloning methods, the GeneMorph II E ZClone domain mutagenesis kit* offers an easy and fast cloning method to perform targeted random mutagenesis on protein domains and promoter elements, while delivering a uniform mutational spectrum. Utilizing a unique cloning method, the GeneMorph II E ZClone kit allows you to target specific protein domains or promoter elements without the need for restriction sites or sub-cloning (see Figure 1).1, 2The kit contains Mutazyme II DNA polymerase, which deliberately introduces mutations during PCR with a more uniform mutational spectrum compared to other error-prone PCR enzymes. Additionally, this kit includes an optimized reaction buffer for the error-prone PCR step so that you need only vary the input DNA amount added to the reaction to produce the desired mutational frequency. The resulting purified mutated PCR products serve as megaprimers for the E ZClone reaction** during which they are denatured and annealed to the original donor plasmid and extended with a specialized enzyme mix containing a high fidelity DNA polymerase. Usinga high-fidelity polymerase minimizes unwanted secondary mutations during the cloning process, which can affect downstream results. The E ZClone reaction is temperature cycled several times before being treated with a unique enzyme to remove parental DNA prior to transformation into competent E. coli . Our XL10-Gold ultracompetent cells are included to maximize library size and diversity. Screening libraries created by random mutagenesis allows researchers to identify beneficial mutations in the absence of structural information, or when such mutations are difficult to predict from protein structure.3* U.S. Patent Nos. 6,803,216, 5,489,523, and patent pending.** U.S. Patent Nos. 6,713,285, 6,391,548, 5,789,166 and 5,932,419, and patents pending.变性的退火亲代在之前F IGURE 1 GeneMorph II EZClone domain mutagenesis kit method.变性退火保真度Eliminate Bias and Easily Control Mutation RateThe mutational bias exhibited by error-prone PCR enzymes undoubtedly skews representation of random mutant libraries, diminishing the effective size of the collection produced by error-prone PCR. Mutazyme II DNA polymerase is a novel error-prone PCR enzyme blend, formulated to provide useful mutation rates with minimal mutational bias. Mutazyme II is a blend of two error-prone DNA polymerases—Mutazyme I DNA polymerase (from the GeneMorph Random Mutagenesis Kit) and a novel Taq DNA polymerase mutant that exhibits increased misinsertion and misextension frequencies compared to wild type Taq . For the Mutazyme II polymerase formulation, the Mutazyme I polymerase and the Taq polymerase mutant have been combined to produce a less biased mutational spectrum with equivalent mutation rates at A’s and T’s vs. G’s and C’s. Therefore, libraries created with Mutazyme II exhibit greater mutant representation compared to libraries generated with other enzymes.With the GeneMorph II E ZClone kit, mutation rates of 1–16 mutations per kb can be achieved using the provided buffer, which is optimized for high product yield. The desired mutation rate can be controlled simply by varying the initial amount of target DNA in the reaction or the number of amplification cycles performed.How Mutation Frequency is ControlledMutation frequency is the product of DNA polymerase error rate and number of duplications (see Appendix ). In the GeneMorph II EZClone kit, a sufficiently high error rate is achieved through use of Mutazyme II DNA polymerase. A low, medium or high mutation frequency is produced by adjusting the initial target DNA amounts in the amplification reactions. For the same PCR yield, targets amplified from low amounts of target DNA undergo more duplications than targets amplified from high concentrations of DNA. The more times a target is replicated, the more errors accumulate. Therefore, higher mutation frequencies are achieved simply by lowering input DNA template concentration. Conversely, lower PCR mutation frequencies can be achieved by using higher DNA template concentrations to limit the number of target duplications. Mutation rates can also be decreased by lowering the number of cycles to achieve fewer target duplications. For targets that produce high product yields after 30 cycles, lower mutation rates can be achieved by amplifying lower target amounts for 20–25 cycles. Selecting the Appropriate Mutation FrequencyThe GeneMorph II EZClone kit allows researchers to choose the mutation frequency that is most appropriate for a particular application. For analyzing protein structure-function relationships, the desired mutation frequency is one amino acid change (1–2 nucleotide changes) per gene.4 In directed evolution studies, mutation frequencies of 1–4 amino acid changes (2–7 nucleotide changes) per gene are commonly employed.5–8 Proteins with improved activities have also been isolated from highly mutagenized libraries exhibiting 20 mutations per gene.3无疑地歪斜地减少经历重复重复Achieving the Desired Mutation FrequencyTable I presents the initial amount of target DNA required to produce low, medium, or high mutation frequencies. An initial target amount of 500–1000 ng is recommended to achieve low mutation frequencies of 0–4.5 mutations/kb. Low mutation frequencies can also be achieved by using 100–500 ng of target DNA with a lower number of PCR cycles (see Cycle Number in Preprotocol Considerations). Initial target amounts ranging from 100–500 ng are recommended for producing mutation frequencies of 4.5–9 mutations/kb (medium mutation frequency range). High mutation frequencies (>9 mutations/kb) are obtained by using 0.1–100 ng of input target DNA, where the highest mutation rates can be achieved using the lowest recommended target amounts. Mutation rates up to 16 mutations per kb have been achieved using 0.01 ng of target DNA, although PCR product yields tend to decrease at amounts below 0.1 ng. The predicted mutation frequencies shown in Table I are accurate for amplification reactions producing the indicated approximate fold amplification. The actual number of mutations in individual clones may differ as the values in Table I represent the average mutation frequency for the entire pool of clones.T ABLE IMutation Frequency vs. Initial Target QuantityMutation rate Mutation frequency(mutations/kb)aInitial targetamount (ng)b,cRecommended foldamplificationLow 0–4.5 500–1000 1.5–10 Medium 4.5–9 100–500 10–100High 9–16 0.1–100 100–10,000 a These values are accurate for reactions achieving the approximate fold amplification (total yield/input DNA) indicated. The actual number of mutations in each clone may differ as these values represent the average frequency for all clones.b The amount of template indicated is the amount of target DNA to be amplified, not the total amount of DNA template to add to the reaction. See Initial Amount of Target in Preprotocol Considerations for an example on how to calculate initial target amount.c The recommended DNA target amounts are higher for Mutazyme II compared to Mutazyme I since Mutazyme II exhibits a ~3-fold higher error rate compared to Mutazyme I.Mutational Spectrum of the GeneMorph II KitThe mutational spectra of Mutazyme II DNA polymerase, Mutazyme I DNA polymerase, and Taq DNA polymerase (with Mn 2+-containing buffer and unbalanced dNTP concentrations) are compared in Table II. These error-prone PCR enzymes introduce all possible nucleotide substitutions, however, Mutazyme II DNA polymerase exhibits less mutational bias compared to Mutazyme I and Taq DNA polymerases.There are several ways to assess bias in an enzyme’s mutational spectrum.Bias can be examined by analyzing the ratio of transition (Ts) to transversion (Tv) mutations produced. Transition mutations are purine (A and G) to purine changes and pyrimidine (C and T) to pyrimidine changes, while transversions are purine to pyrimidine and pyrimidine to purine changes. There are eight possible transversions and four possible transitions, and an enzyme completely lacking bias would exhibit a Ts/Tv ratio of 0.5. Secondly, mutational bias has been assessed by calculating the ratio of AT →GC to GC →AT transition mutations (AT →GC/GC →AT ratio), which would equal 1 for a completely unbiased enzyme. Thirdly, mutational bias can be assessed by comparing the frequency of mutating A’s and T’s vs. the frequency of mutating G’s and C’s (AT →NN/GC →NN ratio), which should be equal for an unbiased DNA polymerase.ll The Taq DNA polymerase was used in the PCR with Mn 2+-containing buffer andunbalanced deoxynucleotide concentrations, which are mutagenic conditions for Taq DNA polymerase.转换范围 频谱颠换T ABLE IIMutational Spectra of Mutazyme and Taq DNA PolymerasesType(s) of mutations Mutazyme IIDNA polymerase aMutazyme IDNA polymerase aTaq DNA polymerase(Reference 5) bBias IndicatorsTs/Tv 0.91.20.8 AT→GC/GC→AT 0.6 0.2 1.9A→N, T→N 50.7% 25.6% 75.9%G→N, C→N 43.8% 72.5% 19.6% TransitionsA→G, T→C 17.5% 10.3% 27.6%G→A, C→T 25.5% 43.7% 13.6% TransversionsA→T, T→A 28.5% 11.1%40.9%A→C, T→G 4.7% 4.2% 7.3%G→C, C→G 4.1% 8.8% 1.4%G→T, C→A 14.1% 20.0% 4.5% Insertions and DeletionsInsertions 0.7%0.8%0.3% Deletions 4.8%1.1%4.2% Mutation FrequencyMutations/kb (per PCR)c3–16 (per PCR) <1 to 7 (per PCR) 4.9 (per PCR)a The Mutazyme DNA polymerases were used with the corresponding GeneMorph random mutagenesis kits.b The Taq DNA polymerase was used with Mn2+-containing buffer and unbalanced dNTP concentrations, which are mutagenic conditions for Taq DNA polymerase.c Initial target amounts of 16 pg to 1 μg (Mutazyme II DNA polymerase), 1 pg to 100 ng (Mutazyme I DNA polymerase), and 0.01 nM template (Taq DNA polymerase) were used to generate data.As shown in Table II, error-prone enzymes generally favor transitions overtransversions, as shown by Ts/Tv ratios greater than 0.5, with Mutazyme IIand Taq exhibiting a somewhat higher tendency to create transversions overtransitions and Mutazyme I exhibiting a greater tendency for introducingtransitions over transversions. E xamining transition mutation frequenciesshows that Mutazyme II produces AT→GC and GC→AT mutations withsimilar rates (AT→GC/GC→AT ratio = 0.6), while Mutazyme I is 4 timesmore likely to generate GC→AT transitions over AT→GC transitions, andTaq is 2 times more likely to introduce AT→GC transitions over GC→ATtransitions. In addition, Mutazyme II DNA polymerase introduces mutationsat A’s and T’s only slightly more frequently than G’s and C’s. In contrast,Mutazyme I is nearly 3 times more likely to mutate G’s and C’s, while Taqunder error-prone conditions is 4 times more likely to mutate A’s and T’sthan G’s and C’s.The spectrum of mutations produced by the GeneMorph II EZClone kit is the same at all mutation frequencies. With the GeneMorph II EZClone kit, low, medium, and high mutation frequencies are achieved using a single set of buffer conditions (MgCl 2, balanced dNTPs) optimized for high product yield. The only parameter varied is the initial amount of target DNA in the reaction or the number of cycles employed. In contrast, Taq DNA polymerase–based mutagenesis methods typically employ different sets of reaction conditions to vary mutation levels. Varying the buffer conditions (e.g., different Mn 2+ concentrations) and/or the concentrations of one or more nucleotides to alter mutation frequency can lead to changes in Taq ’s mutational spectrum and increased mutational bias.Furthermore, mutational hotspots have not been observed in any of the mutagenized genes generated by Mutazyme II DNA polymerase that have been sequenced.9EZClone ReactionThe E ZClone reaction utilizes (1) a supercoiled double-stranded DNA (dsDNA) vector containing the same region targeted for mutagenesis in the mutagenesis reaction and (2) two megaprimers generated in the mutagenesis reaction containing random mutations. The E ZClone enzyme mix is a 2× formulation containing a high fidelity DNA polymerase to minimize unwanted second site errors, an optimized PCR reaction buffer, magnesium, and dNTPs. The megaprimers, each complementary to opposite strands of the vector, are extended during temperature cycling by the EZClone enzyme mix, without primer displacement. Extension of the megaprimers generates a mutated plasmid containing staggered nicks. Following temperature cycling, the product is treated with Dpn I restriction enzyme. The Dpn I endonuclease (target sequence: 5´-Gm 6ATC-3´) is specific for methylated and hemimethylated DNA and is used to digest the parental DNA template. Because DNA produced during PCR is not methylated, Dpn I selects for the mutation-containing synthesized DNA.10 The nicked vector DNA incorporating the desired mutations is then transformed into competent cells (provided), where the nicks are repaired by endogenous enzymes in the cell. Note While plasmid DNA isolated from almost all of the commonly usedE. coli strains (dam +) is methylated and is a suitable template formutagenesis, plasmid DNA isolated from the exceptional dam – E. coli strains, including JM110 and SCS110, is not suitable. 超螺旋的突变易发生的大引物变异发生高保真充分利用的镁充足的延伸缺口甲基化半甲基 消化亲本带切口的意外的G ENE M ORPH II EZC LONE D OMAIN M UTAGENESIS C ONTROLTo demonstrate the effectiveness of the GeneMorph II EZClone method the3.0-kb positive control plasmid, which contains the lacZ gene, is used to testthe efficiency of mutant megaprimer synthesis and the E ZClone reaction.XL10-Gold ultracompetent cells* transformed with this control plasmidappear blue on LB–ampicillin agar plates (see Preparation of Media andReagents), containing IPTG and X-gal. Following random mutagenesis andE ZCloning, >25% of the colonies plated appear white on LB–ampicillinplates containing IPTG and X-gal because β-galactosidase activity has beenobliterated.XL10-G OLD U LTRACOMPETENT C ELLSStratagene XL10-Gold ultracompetent cells, a derivative of the highest-efficiency Stratagene competent cell line, XL2-Blue MRF´, possess the Htephenotype, which increases transformation efficiency of ligated DNA.11XL10-Gold cells are both endonuclease deficient (endA1) andrecombination deficient (recA). The endA1 mutation greatly improves thequality of plasmid miniprep DNA,12 and the recA mutation helps ensureinsert stability. In addition, the McrA, McrCB, McrF, Mrr, and HsdRsystems have been removed from XL10-Gold ultracompetent cells. ThemcrA, mcrCB and mrr mutations prevent cleavage of cloned DNA thatcarries cytosine and/or adenine methylation, which is often present ineukaryotic DNA and cDNA.13–15 The McrA and McrCB systems recognizeand restrict methylated cytosine DNA sequences, and the Mrr systemrecognizes and restricts methylated adenine DNA sequences. The Mrrsystem also restricts methylated cytosine DNA sequences with a specificitydiffering from that of McrA and McrCB. This activity has been namedMcrF. This McrF activity against methylated cytosines has been shown tobe equal to or greater than the restriction activity of the McrA and McrCBsystems.16 The hsdR mutation prevents the cleavage of cloned DNA by theEco K (hsdR) endonuclease system. XL10-Gold cells grow faster than XL1or XL2-Blue cells, resulting in larger colonies. To permit blue-white colorscreening, the XL10-Gold ultracompetent cells contain the lacI q ZΔM15 geneon the F´ episome.Host strain References GenotypeXL10-Gold ultracompetent cells 11, 17, 18 Tet RΔ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte[F´ proAB lacI q ZΔM15 Tn10 (Tet R) Amy Cam R]It is important to store the XL10-Gold ultracompetent cells at –80°C to prevent a loss of efficiency. For best results, please follow the directions outlined in the following sections.* U.S. Patent Nos. 6,706,525, 5,512,468 and 5,707,841 and patents pending and equivalentforeign patentsP REPROTOCOL C ONSIDERATIONSMutant Megaprimer Synthesis ConsiderationsAmplification TargetsThe GeneMorph II EZClone kit has been used to mutagenize targets up to3.5 kb in length from plasmid DNA.Genomic DNA templates are not generally recommended for error-pronePCR as researchers are limited to medium-to-high mutation levels due to thelow copy number of genomic DNA targets. If genomic DNA is the onlysource of the target gene, we recommend amplifying the target with ahigh-fidelity DNA polymerase, such as PfuUltra high-fidelity DNApolymerase, followed by re-amplification of the diluted PCR product withMutazyme II DNA polymerase.Initial Amount of Target DNAThe mutation frequency depends upon the initial amount of target DNAemployed in the reaction. The amount of target to add to a reaction can bedetermined using Table I.The initial amount of target DNA required to achieve a particularmutation frequency refers to the amount of target DNA to amplify, notthe total amount of plasmid DNA template to add to the reaction. As anexample, to mutagenize a 1.0-kb target gene at a low mutation frequency, aninitial target amount of 500 ng is recommended. For a 1.0-kb target genethat is an insert in a 3.0-kb plasmid (the total construct is 4.0 kb), 2 μg of theplasmid construct should be added to the reaction to provide 500 ng of targetDNA.Cycle NumberIn addition to using higher target DNA amounts, mutation rates can also be lowered by decreasing the number of cycles employed to achieve fewertarget duplications. For targets that produce high product yields after30 cycles, lower mutation rates can be achieved by amplifying lower target amounts for 20 to 25 cycles (see Table III).T ABLE IIIAchieving Low Mutation Frequency Using Fewer Cycle Numbers Mutation frequency(mutations/kb) a Cycle Number Initial target amount b0–4.5 (low range)ng20–25 100ngng–100030 500a These values are accurate for reactions achieving the approximate 1.5–10 fold amplification (total yield/input DNA). The actual number of mutations in each clonemay differ as these values represent the average frequency for all clones.b The amount of template indicated is the amount of target DNA to be amplified, not thetotal amount of DNA template to add to the reaction. See Initial Amount of Target in Preprotocol Considerations for an example on how to calculate initial target amount. Primer DesignStandard PCR primers flanking the region targeted for mutagenesis are usedin the mutant megaprimer synthesis reaction. For best results, PCR primers should be designed with similar melting temperatures ranging from 55 to72°C. The use of primers with melting temperatures within this rangereduces false priming and ensures complete denaturation of unextended primers at 94–95°C.PCR Product YieldThe PCR product yield should be within the recommended range to obtainthe predicted mutation frequencies listed in Tables I and III. To ensuresufficient product yield, sample PCR reactions are electrophoresed adjacentto a DNA standard provided in the kit. PCR product yields are quantified by被量化comparing the staining intensity of PCR product bands to the DNAstandard.Achieving High Mutation FrequenciesThe highest mutation frequency that can be achieved in one round of PCR islimited by the minimum amount of target DNA that can be amplified in high product yield. In the GeneMorph II kit, we recommend using 0.1–100 ng of target DNA, which is sufficient to produce high product yields after30 cycles and mutation frequencies up to 9–16 mutations per kb of target. Higher mutation frequencies can be achieved by amplifying from<0.1ng target DNA, although product yields may be noticeably lower.Alternatively, mutation frequencies > 20 mutations per kb can be achievedby performing sequential PCRs, in which a small aliquot of the first PCRreaction is re-amplified in a second PCR reaction.EZClone Reaction ConsiderationsRequired Host StrainE nsure that the plasmid DNA template is isolated from a dam+ E. colistrain. The majority of the commonly used E. coli strains are dam+.Plasmid DNA isolated from dam– strains (e.g. JM110 and SCS110) is notsuitable.Plasmid DNA Template GuidelinesThe plasmid DNA template used in the EZClone reaction may be the sameor different than the original plasmid DNA used as template in the mutantmegaprimer synthesis reaction, provided that the region targeted in themutagenesis reaction is present in the plasmid.The plasmid DNA template used in the E ZClone reaction should be lessthan 10 kb in length to ensure full extension during temperature cycling.P ROTOCOLMutant Megaprimer SynthesisNote Gently mix and centrifuge each component before use. Prepare allreactions on ice.1. Refer to Table I to determine the initial amount of target to use in eachreaction.Note Target DNA refers to the DNA sequence to be amplified,not the total amount of plasmid DNA in the reaction(see Initial Amount of Target in PreprotocolConsiderations).2. Prepare the control reaction as indicated below:5 μl of 10× Mutazyme II reaction buffer2 μl of 0.5 ng/μl positive control plasmid (dilute the controlprovided 1:20 in high-quality water for a final concentrationof 0.5 ng/μl)1 μl of positive control primer mix (125 ng/μl of each primer)1 μl of 40 mM dNTP mix (200 μM each final)40 μl of ddH2O1 μl of Mutazyme II DNA polymerase (2.5 U/μl)3. Prepare the sample reaction(s) as indicated below:5 μl of 10× Mutazyme II reaction bufferx μl template (see Table I for recommended amount)1 μl of sample primers (125 ng/μl of each primer)1 μl of 40 mM dNTP mix (200 μM each final)x μl of ddH2O for a final reaction volume of 50 μl1 μl of Mutazyme II DNA polymerase (2.5 U/μl)4. Centrifuge each reaction briefly.5. If the thermal cycler does not have a heated lid, overlay each reactionwith a few drops of mineral oil.。